The epidermal growth factor receptor (EGFR) is overexpressed in many human cancers and cancer cell lines due to gene amplification and/or increased gene transcription. We have continued our efforts to identify and characterize transcription factors that regulate EGFR gene expression.In our studies of EGFR gene regulation, we have documented the interaction of transcription factors such as Sp1, AP2 and p53 with the promoter region. Upon further examination of the EGFR promoter sequence, eleven potential AP1 binding sites were indicated. To test the ability of the EGFR promoter region to bind AP1, DNase I footprinting analysis was performed. Seven regions in the 1.1 kbp promoter were shown to be capable of binding AP1. To examine whether AP1 could effect changes in EGFR expression, cotransfection experiments were performed using a c-Jun expression vector and EGFR reporter constructs. The EGFR promoter activity was enhanced 7-fold by c-Jun. Jun B was also able to enhance EGFR expression. However, c-fos was not. We have also shown that a dominant-negative expression construct, A- Fos, which has been reported to inhibit c-Jun activation, prevents c- Jun activation of EGFR promoter activity. A c-Jun construct lacking the transactivation domain also fails to increase EGFR promoter activity. Moreover, we have shown that MCF-7 cells that are stably overexpressing c-Jun have an increase in EGFR level. Also, an ovarian cancer cell line that is cisplatin resistant has been shown to have higher AP1 activity than the parental cisplatin-sensitive cell line. We have shown that the cisplatin resistant cell line also has an increased level of EGFR as compared to the parental cell line. These results provide evidence that AP1 plays a role in upregulating EGFR expression. As part of our ongoing effort to further the understanding of EGFR gene regulation, we collaborated with Gordon Guroff?s laboratory (Section on Growth Factors, NICHD). We were able to document an inverse correlation of EGFR downregulation and increased GCF2 expression in PC12 cells that were treated with nerve growth factor.Another factor that we have shown to interact with the EGFR promoter region is the transcriptional repressor GCF2. To learn more about the GCF2 gene, we isolated a PAC clone that hybridizes with the GCF2 cDNA. Genomic DNA from this clone was labeled and used in FISH analysis to determine chromosomal location. The GCF2 gene was localized to chromosome 3 q27 region. This is a region that is frequently overrepresented in malignant lymphomas. This is consistent with our finding of high level GCF2 expression in six Burkitts lymphoma cell lines and in one cell line derived from a T- cell lymphoma.In collaboration with Levon Khachigan, University of New South Wales, Sidney Australia, we also showed that the platelet-derived growth factor A-chain (PDGF-A) promoter was repressed by GCF2. The binding site was determined by DNase I footprinting analysis and found to be identical in sequence to the site in the EGFR promoter. The GCF2 binding site in the PDGF-A promoter overlaps binding sites for Sp1, Sp3 and Egr-1. GCF2 was shown to compete with these three factors for binding to this site.To determine the regions of GCF2 necessary for binding DNA and repression of promoter activity, we prepared deletions of GCF2. Deletions that contained amino acids 1-510 were not able to bind DNA or repress promoter activity. Deletion mutants that contained amino acids 1-528 could bind DNA and repress promoter activity. The region between amino acids 510 and 528 is very basic and contains a sequence that is similar to a nuclear localization sequence. To determine the cellular location(s) of GCF2, we prepared nuclear and cytoplasmic fractions from cells and used western blotting to locate GCF2. GCF2 was found in both nuclear and cytoplasmic compartments of the cell. The percentage of GCF2 in each compartment was cell line dependent. Thus, GCF2 is a DNA binding protein that represses promoter activity and may compete with transcriptional activators to mediate this effect. - activation, expression, factors, promoter, regulation, repression, Transcription, - Neither Human Subjects nor Human Tissues